A subset of Toll-like receptors (TLRs) recognizes the nucleic acids of viral and bacterial genomes. This recognition strategy can come at a significant cost, as these types of nucleic acid are also present in the host. Most of the time, recognition of self nucleic acid is avoided. In some instances, though, TLRs contribute to a vigorous anti-nucleic acid immune response called systemic lupus erythematosus (SLE). This proposal focuses on defining the mechanisms that prevent recognition of self nucleic acid while still allowing detection of foreign nucleic acid. We are addressing this fundamental issue using TLR9 as a representative example of the group of nucleic acid-specific TLRs. TLR9 recognizes unmethylated CpG motifs in double-stranded DNA and has been shown to play a role in the immune response to a number of double-stranded DNA viruses. TLR9 has also been implicated in the pathology of SLE. An intriguing aspect of TLR9 biology is that it does not traffic to the cell surface, but instead localizes to and recognizes ligand within intracellular compartments. We have evidence that the cell biology of TLR9 is regulated at multiple levels. This research application aims to define the mechanisms that regulate TLR9 localization within the cell. In Aim 1 we will define the region within TLR9 necessary for its intracellular retention. In Aim 2, we will use novel biochemical techniques to characterize the pathways that control TLR9 localization and trafficking. Finally, in Aim 3 we express mutant receptors in mice and test the hypothesis that intracellular localization is necessary to avoid self DNA recognition. Collectively, these Aims will address how the cell biology of TLR9 is regulated and will test the importance of this regulation in maintaining proper self/non-self discrimination by this receptor. PUBLIC HEALTH RELEVANCE: This proposal deals with the balance that must be maintained by the immune system between recognition of infection and prevention of autoimmunity. The work focuses on TLR9, a protein that normally recognizes viral DNA, but can sometimes inappropriately recognize self DNA and cause autoimmune disease. The goal of this research is to understand how TLR9 recognizes viral DNA while avoiding self DNA.